Recently I did RNA sequencing using ovarian tissue samples to find out the differencial gene expression in ovary based on WT and mutant gene. Bioinformatics results analysis show one of the gene expression is 0. However, when I do qPCR for the same gene I can see upregulation of that gene in several folds and I confirm this results by repeating the qPCR. Now I am confusing, why RNA seq data show no expression of such gene and my qPCR show upregulation of the same gene. The given gene is having one transcript. Can anybody explain me why this is happening?
In my expression false zero expression of genes happens most commonly if haplotypes are considered whereby multiple (almost) identical version of the genes will be quantified - and since old school (aka on the verge of obsolete) methods only counts uniquely mapped genes there will be zero counts.
As a quick sanity check get the bioinformatician to just extract reads that overlap the genomic region of interest to see if there are any. Should be very easy from the bamfile(s) via a command like this:
samtools view yourBamFile.bam chr1:3052830-3052840
Where the coordinates (chr : start - end ) should be swapped for your gene of interest. If this works the bioinformatician would need to requantify the data taking haplotypes into account and then you should also try convincing her/him to switch to more modern quantification ways - you can read more about them (and why to use them) here.